Activation mechanisms of voltage-gated K+ and Na+ channels

电压门控离子通道是一种重要的膜蛋白，通过膜上电位的变化来激活和失活。电压门控离子通道的激活机制仍然不清楚，部分原因是在膜电位存在的情况下，缺乏关于静息状态下通道的高分辨率结构信息。了解静息状态下电压门控离子通道的结构将有助于理解它们的激活机制。在这里，我建议使用膜蛋白生物化学、生物物理学和亚断层图像平均相结合的方法，在高分辨率的受控膜电位下获得闭合脂泡中电压门控离子通道的结构信息。具体地说，我们的目标是确定三种不同电压门控离子通道在静息状态下的结构：(1)细菌电压门控钠通道NaChBac，(2)传统哺乳动物电压门控钾通道Kv1.2和(3)具有非交换结构域的非传统哺乳动物电压门控通道。实验方法包括蛋白质的异源表达、纯化、重组为脂质体、生物物理表征和结构分析。为了成功地进行生物解释，从低温电子断层图像中求取亚断层图像的高分辨率是至关重要的，而低温电子断层图像是原位结构分析的首选方法。对于这种方法，将进行优化图像处理工作流程的开发。我们将使用计算机视觉领域的现代方法来执行具有各向异性分辨率的噪声3D体积的更精确的配准。我们将进一步说明亚断层图像平均数据处理中典型的几何和电子光学失真。总而言之，这些方法的发展将使我们能够达到α螺旋甚至接近原子的分辨率。所开发的亚断层图像平均方法将是通用的；它们将被公开发布，并由其他研究人员和我们用于在膜电位下确定闭合脂泡中离子通道的结构。了解静息状态下电压门控离子通道的结构将大大加深我们对电压门控的分子机制的理解，这将为开发更具选择性的治疗多种心脏和神经疾病的药物奠定基础。

Voltage-gated ion channels are critically important membrane proteins that are activated and deactivated by changes in electric potential across membranes. The activation mechanism of voltage-gated ion channels remains elusive, in part due to lack of high-resolution structural information on the channels in resting state in the presence of membrane potential. Knowledge of the structure of voltage-gated ion channels in resting state will allow understanding their activation mechanisms. Here, I propose to use a combination of membrane protein biochemistry, biophysics, and subtomogram averaging to obtain structural information on voltage-gated ion channels in closed lipid vesicles under a controlled membrane potential at high resolution. Specifically, we aim to determine the resting state structures of three different voltage-gated ion channels in the resting states: (1) a bacterial voltage-gated sodium channel NaChBac, (2) a conventional mammalian voltage-gated potassium channel Kv1.2 and (3) a non-conventional mammalian voltage-gated channel with non-swapped domains. Experimental approach involves heterologous expression of the the proteins, their purification, reconstitution into liposomes, biophysical characterization and structural analysis. In order to succeed in biological interpretation, it is critical to achieve high resolution with subtomogram averaging from cryo electron tomograms which is the method of choice for structural analysis in situ. For this method developments optimizing the image processing workflows will be performed. We will use modern approaches from the computer vision field in order to perform more precise registration of noisy 3D volumes with anisotropic resolution. We will further account for the geometrical and electron optical distortions typical to processing of subtomogram averaging data. Taken together the method developments will allow reaching alpha-helical or even near-atomic resolutions.The developed methods for subtomogram averaging will be of general use; they will be publically released and used by other researchers and by us for determining the structures of ion channels in closed lipid vesicles under the membrane potential. Knowledge of the structures of the voltage-gated ion channels in their resting states will significantly further our understanding of the molecular mechanisms of voltage gating which will serve as a basis for the development of more selective drugs against multiple cardiac and neurological disorders.